Many of the procedures commonly used in molecular biology involve the generation and analysis of blots from DNA, RNA, protein samples, or other biological components. These blots consist of thin sheets of nitrocellulose, charged nylon, or the like, to which the samples species to be studied are bound by any of a number of procedures. The sheets provide support for the sample species and allow the researcher to visualize and analyze the species of interest in a more convenient, albeit somewhat cumbersome, manner.
The current techniques used to generate and analyze these blots have been largely adapted from other procedures, and little effort has been made to create an efficient system to support these activities in a more dependable fashion. In the classical procedure, the sample to be studied is first separated on the basis of its size using gel electrophoresis. The sample is then driven out of the gel and onto the nylon or nitrocellulose membranes (transfer membranes) by fluid flow (Southern, E., J. Mol. Biol. 98:503, 1975) or by electrical current (see, e.g., Kreisher, J. H., U.S. Pat. No. 4,589,965, 1986). These procedures are collectively called blotting and the resultant membrane with the sample species affixed to it is called a blot. Usually the blot is then heated or treated with ultraviolet radiation or other means to cause the sample species to bind covalently to the membrane support matrix. The blot is then ready to be used in the study of the species that has been attached to it.
In conventional blotting procedures, unsupported transfer membranes are first layered on top of the gels by hand, and then the gel and membrane together are sandwiched in the blotting apparatus. Since these steps are performed manually, it is difficult to place an unsupported membrane upon a gel accurately to insure even or uniform transfer of the material from the gel to the membrane. It is even more difficult to duplicate membrane placement accurately and consistently from gel to gel. This problem results in a good deal of variability in the resulting configuration of the blots, making it difficult to compare successive blots directly. In addition, these prior art techniques are time-consuming, requiring at least 12-14 hours for capillary transfer and 2-4 hours for electroblotting. Electroblotting also requires costly power supplies and transfer chambers, as well as large quantitites of buffer.
The next step in the analysis of the blots, called hybridization or visualization, uses a probe to bind to certain select species on the blot. The probe is labeled, usually with radioisotopes, so that desired fragments can be imaged subsequently to give a permanent record of the results. In the commonly used procedures, the blots are placed in plastic bags, and the radioactive probe solution is added. All air bubbles are removed by hand; then the bag is heat-sealed. The bag is then incubated in a water bath for an appropriate time, after which the blot is removed and rinsed several times to remove any probe that has not bound to its specific target species. These steps are labor-intensive and cumbersome (the researcher must work behind a safety shield), and inevitably result in contamination of the surroundings with radioactivity.
Certain commercial products have been introduced to carry out hybridization with greater facility. Hybrid-Ease.TM. (Hoefer Scientific Instruments, San Francisco, CA) and Turbo-Blot.TM. (American Bionetics, Malvern, PA) are examples of such devices. While they are significantly beter than the sealable plastic bags, each has certain shortcomings. Hybrid-Ease has a very large incubation chamber, that requires 3-6 times the volume of probe solution required by the plastic bags. The probes are very costly and must be at fairly high concentation in the hybridization solution; this is a serious drawback. The Turbo-Blot unit is little more than a plastic bag with tubing fittings to introduce the solutions and clamps to seal the bag. It is complicated to set up and use, and also requires considerable volumes of probe solution. Neither of these units is designed to work in conjunction with or configured to facilitate transition from preceding to successive steps in the generation and analysis of the blots. That is, there is no convenient blot support means, blot frame structure, blot handling apparatus or smooth sequential procedure for accomplishing and facilitating successive steps.
Next the bound, radiolabeled probe on the blot is imaged by placing the blot against an x-ray film for a period of 10-72 hours. Since it is not often possible to ascertain before-hand how much radioactivity has bound to the blot, it is not possible to pre-determine the correct exposure time. It is thus common prior art practice to use two x-ray films, one on either side of the blot. This "sandwich" is taped onto the inside of a light-tight container. After an appropriate time, the outer film is removed and developed. If it is not fully exposed, the second film is left in place for an additional period of time. However, during the removal of the first film the blot is often shifted in relation to the second film, causing a blurred image. This cumbersome process is made more difficult because of the fact that these steps of necessity are performed in a darkroom and the blot itself is really just a thin piece of membranous material and is therefore difficult to handle. The problem is exacerbated when one realizes that the radioactivity itself is decreasing rapidly because of the natural halflife of the isotopes. For example, the most commonly used radioactive isotope, phosphorus 32, has a halflife of only 14 days. Thus, if the imaging step has to be repeated, the amount of isotope that is present and the amount of time required to detect this radioactivity are both different from that observed the first time.
Finally, even though prior art blots may be reused a number of times, they are difficult to store because they are quite susceptible to contamination (one fingerprint will often hold more DNA than resides on an entire blot) and physical damage. It is difficult to protect these membranes from such contamination since the contaminants are rather ubiquitous, so that any storage package will likely have in it significant levels of contaminants. In addition, certain types of membranes become brittle with use, making them very fragile and difficult to handle without peripheral support. For these reasons, storage of the blots in most conventional media such as paper or plastic envelopes or boxes is often unsatisfactory.